【摘要】 环二鸟苷酸（bis-（3’-5’）-cyclic dimeric guanosine monophosphate, c-di-GMP）是广泛存在于细菌中的第二信使分子,可通过STING （stimulator of interferon genes）信号通路参与先天免疫应答,是一种潜在的疫苗佐剂。为明确其先天免疫调节机制和评估免疫佐剂效果,c-di-GMP的高效合成急需解决。为建立一种相对简单、高效的c-di-GMP生物合成方法 ,实现体外制备c-di-GMP,本研究构建大肠杆菌（Escherichia coli）二核苷酸环化酶（dinucleotide cyclase V, DncV）重组表达载体,经异丙基-β-D-硫代半乳糖苷（isopropy-β-D-thiogalactoside, IPTG）诱导表达和His亲和纯化获得目的蛋白DncV;利用超高效液相色谱（ultra performance liquid chromatography, UPLC）检测DncV重组蛋白体外催化GTP生成c-di-GMP的反应。研究结果表明,IPTG诱导型表达载体pET-28a-His-DncV构建成功,亲和纯化获得的DncV重组蛋白纯度达到92%;该重组蛋白能够通过酶促反应一步生成c-di-GMP;金属离子对该酶活性影响显著,其中3种金属离子(Mg²⁺, Mn²⁺, Co²⁺)可提高催化效率,另外7种金属离子(Cu²⁺, Zn²⁺, Ni²⁺, Mo²⁺, Bo²⁺, Fe²⁺, Ca²⁺)抑制酶促反应。本研究建立了E. coli DncV重组蛋白催化合成c-di-GMP的方法体系,为c-di-GMP的大规模制备和功能研究提供了基础资料。更多还原

【Abstract】 Bis-（3’-5’）-cyclic dimeric guanosine monophosphate（c-di-GMP） is a second messenger molecule widely present in bacteria and can participate in innate immune response through stimulator of interferon genes（STING） signaling pathway and is a potential vaccine adjuvant. In order to clarify its innate immune regulation mechanism and evaluate the effect of immunoadjuvant, the efficient synthesis of c-di-GMP needs to be solved urgently. In order to establish a relatively simple and efficient c-di-GMP biosynthesis method to prepare c-di-GMP in vitro, this study constructed Escherichia coli dinucleotide cyclase（DncV） recombinant expression vector, and the target protein DncV was obtained by IPTG（isopropy-β-D-thiogalactoside）-induced expression and His affinity purification; Detection of c-di-GMP from GTP catalyzed by DncV recombinant protein by ultra performance liquid chromatography（UPLC） in vitro. The results showed that the IPTGinducible expression vector pET-28 a-His-DncV was successfully constructed, and the purity of the recombinant protein obtained by affinity purification was 92%. The recombinant protein could generate c-diGMP in one step by enzymatic reaction; Metal ions had a significant effect on the activity of the enzyme.Three kinds of metal ions(Mg²⁺, Mn²⁺, Co²⁺) could improve the catalytic efficiency, and the other 7 metal ions(Cu²⁺, Zn²⁺, Ni²⁺, Mo²⁺, Bo²⁺, Fe²⁺, Ca²⁺) inhibited the enzymatic reaction. This study established a method for the synthesis of c-di-GMP by E. coli DncV recombinant protein, which provides basic data for the large-scale preparation and functional study of c-di-GMP.更多还原